CA1137078A - Modified grass pollen antigens - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Oxidative rearrangement and cleavage of hydroxy aromatic rings of quercitin moiety of Timothy grass pollen antigen or antigen fragments gives rise to a modified anti-gen having no antigenic properties but retaining capability of activating T-cells for regulation of immune response.

Description

37~
BACKGROUND OF TEIE INVENTION
, -- :
Recent studies have resuited in a characterization of the antigenic determinant oE antigen B of Timothy grass pollen. The antigen is composed of a flavanoid pigment, quercitin; a disaccharide, cellobiose; and a polypeptide "tail". Anti~en B is the generic name for mixtures containing two or more antigen fragments conventionally termed "antigen D, Dl, D2, D3, etc." Ihey differ from each other in the length of the polypeptide "tail". In anti~en D3, the "tail" consists of a single aminoacid, namely, threonine.
See Malley et al, Dev. Biol.~ Stand., 1975~ 29 (Int. WHO-IABS, Symp. Stand. Control ~llerqens Adm, Man., 1974~ r ~29~40j Kramer-Xleinert et al, Dev. Biol. Stand., 1975r 29 ~Int. WHO-IABS, Symp. Stand. Control Aller- gens Adm~ ~an. r 188-~6; Malle~ et al, Immunochemistry, 1975, Vol. 12, pages 551-554; Malley et al, Journal of ~llergy, Vol. 43, ~lo. 2, February, 1369, -. i _ . . - : .
pages 59-64; Gerard et al, I_munochemis~ 75, Vol. 12 pages 545-9; MallQy et al/ Journa;l of Immunology, Yol~ 99, ` No. 4 (1967), pa~es 825-830, Sanderson et al~ Immunolo~y, .
Uol. 20 ~1971~, pages 1061-1065; and Axen et al, Natu~re Vol. 214 (1967), pages 1302~1304.
In copending Canadian application Serial No 288,312, filed October 7, 1977! no~ Canadian patent 1!099,258 it is disclosed that the various ~ntigen D fragments coupled to various proteins or peptides through the free carbQxyl group of threonine initiate signi~icant histamine reIease from sensitized tissue. It is urther disclosed thereinr however, that these same antigen D fragmen~s coupled to various proteins and petides through the sugar moiety ~ive ;- 30 rise to a dose-dependent inhibition of histamine release from sensiti7ed tissue.

These studies intimate strongly that quercitin represents the major portion of the antigenic dete m inant of c g/~1 ' ' :

137~78 antigen B. ;
Antigen B and its various fraqments may be represented by the structural formula~
O ~0~

H O ~ G - G - ~ - T

wherein G repxesents glucose and T represents threonine or a peptide l~nked to said structure through the threonine mole~
cule.
Antigen Dl has the structure set orth above ~wherein the peptide tail~is such that the molecular weight is about 5,000. Antigen-D2 has a molecular welght~ of about

2~500~ In antigen D3j T is threonine alon The quercitin fraction of the antigen structure is linked to the~glucose moleties vla an~ether linkage through ;
OH groups on the respectlve molecules. The threonine linkage to the glucose moLety is also an ethe~ linka~e through an OH
group on the cellobiose fraction and the OH group of threonine.
The nature of the allergic reaction or response in humans and animals~is not oompletely understood. It is theorized that the allergic response to a foreign antigen, : ~
such as the antigen o;f grass pollen, in~olves the in~eraction of at least two different cell types. One of these is~oonven~
; tionally tarmed a T-cell, or Thymus derived lymphocyte, and the second a B-cell, whi~ch is a bone marrow derived lymphocyte. - `
Each of these two cells, the T ana the B lymphocytesj recognize diffarent parts of the protein that causes the ~llergic : .
responseO The B-lymphocyte reoognizes the antigenic de ~-terminant whereas the T-lymphocyte reco~nizes a different portion of the protein, termed a carrier determinant. To achieve suppression o the immune response involved in allergic diseases one can attack the problem by either sup-:
. ~ , cg/~
--- . ;

3~8 pressing the iormati.on of antibody by blocking B-cell dif-f~renti~tion or by affecting the T-cell population. The B-lymphocyte actually produces the anti~ody~ T-cells do not produce the circulating ~ntibody tllat is involved in allergic disease, but regulate their production. One sub-population of T cells which assists in making antibodies are the so called helper T-cells. They effectively cooperate or :
: interac~ with the B-lymphocyte to produce antibodies ! Another sub-population of T-cells, the "suppressor" T-cell or Ts c~ll, effecti.vely suppresses the action of helper T-cells so ~ ;
that they cannot participate with B-lymphocytes to produce antibodies.
It is an object of t~e present invention to provide a method for modifying the antigenic properties of Timothy antigen B and/or its various fragments to yield a product ~ ;
devoid of antigenic properties but capable of activa-ting T-cells. ~ .
:i: SUMMARY OF THE INVENTI0~7 ~ The present invention is predicated on ~he dis~
:~ covery that s:ub3ecting the quercitin moiety of grass pollen antigen having the structure:

:. OH
HO

HO ~ O - G - G - a ~ T
~;;
: wherein: G represents glucose. and T represents threonine or a peptide linked to the structure through a thxebnine mole~
.: cule to oxidative conditions such tha-t the dihydroxy aromatic rings of the quercitin moiety undergo rearrangement and ring scission yield a ~roduct substanti~lly dev~id o antigenic properties but capable of activating TYcells and inducing s cells.

BRIF,F DESCRIPTION OF' THE DRAWINGS
_ , ~: FIGS. 1~4 represent graphic illustrations of the - 3 _ cg/~

L3 70~8 - ~ ~
antigenic properties of the oxidatively modified antigen of the present invention.
DETAILED DESCRIPTION OF THE INVE:NTION
... . _. . . . . . . . ~
The method of the invention is applicable to any : quercitin moiety containing anti~en or anti~en ~ragment such ~
as antigen B, antigen D, antigen D1, D2, D3, etc. ~ ~:
The method gives rise to a mod.ified antigen which may be compounded in unit dosage form to provlde an ~ ;
immunotherapeutic composition for inhibiting allergic .
reactions in.humans and animals sensitive to the said antigens. :
~-~ It is known that hydroxy aroma-tic rings are sub~
ject to oxidative scission following rearxangement thereof, depending upon the reaction conditions. It is also known ~ ~
that certain oxidations affect a variety.of ammino acids in ;' different ways. It is known that tyrosine and'methionine are present in the polypeptide "tail" of antigens B or D. I-t is~ --further known that oxidative conditions suf~iciently rigorous ~' to effect ring scission in the querci.tin moiety will also ~ ..
.
affect the ~tyroslne and methionine content o~ the polypeptide .~ .

-- 20 "~ail". It has;been found, however, that modi$icatlon of these two.residues and a minimum of other ammino~cid residues ~:

; present in the tail will not affect the capabilit~ of the .: . ~
modified antigen to induce active T-cells~ ::
. Any oxid'ation reaction capable o~ effecting rearrangement and ring scission of the hydroxy aromatic rin~s ~' of the quercitin moiety, while not substantially modifying :
the polypeptide "tail", will result.in a product having.sub- ~;
stantially no antlgenic properties but retainin.g its capability ~ -of activating T-cells and inducing ~s cells. .
; 30 Photooxidative cleavage of the hydrvxy aromatic :
rings has been found to ~e highly effective in producing the :: modified antigen of the~invention. Obviously, the rate of ':~
oxidation and the residues affected depend upon may vari- ' ~
: '

- 4 cg/~
- . - ~ , ~

~ ~ 3~78 ables, including the pH, concentration of the photosensitizer, the size of thc vessel and the intensity and duration of exposure of the sample to light~
It is to be understood, however; that any~oxidative procedure which results in rearrangement and scission of the hydroxyaromatic rings of quercitin in the antigen or fragments thereof while avoiding substantial modification of the peptide -tail may be'employed according to the present invention.
Thus, chloramine-T ~sodium para-toluenesulfon-chloramine], lactoperoxid se and potassium permanganate may also be employed as suitable oxidizing agents for modification ~;
of the quercitin moiety of the antigen or antigen fra~ments.
EXAMPL~ I
Timothy pollen extract (WST) and purified antiqen B, AgB, wexe prepared as described by Malley et~alt ~
Immunol 99, 825 (1967). The protein concentration of these ~' antigen preparations was determined by nesslerization .
; (ampbell et al~,~Me~thods__in Immuno'l'ogy, Ed. W.A. Benjamin, N.Y., pg. 53, 1963)o ~he AgB was treated with 8 M urea, as described by ~, ~
Takatsu and Ishizaka (Cell Immunol., 20, 276, 1975), i.er, two grams of~urea were added to 2.5 ml of a 1% solution of ~gB in 0.1 M Tris-HCl buffer (pH 8.0). Ater 18 hrs. of incu-bation at room temperature, the protein solution was dialyzed against phosphate-buffered saline, concentrated back to its '-;
original volume (2~5'ml) by negative pressure,~ and tested fQr its reagin~binding activity by the radioallergosorbent (RAST) . .
înhibition method. ~-' 30 Photooxidation of Anti~en B
Three milligrams of AgB (0~4 ml) and 2 ml of 0.2 M
phosphate bu~fer (pH 9) were added to 10-ml serum ~ials~ The vials were covered with aluminum foil so that no light would !~ .
~'f3~ ~

cg/~
' get in. One-half millimeter of methylene blue photo-sensitizer 0.2 mg/ml in 0.2 M phosphate buffer (pH 9), was added to each vial, and the reaction mixture was saturated with 2 by a gentle bubbling of the gas into the solution for S to 10 min. The serum vials were stoppered immediately and incubated at 37 C for 30 min. The aluminum foil was removed from the vials, and their contents were exposed to two 150-watt lights positioned 1.5 inches from the vials for varying periods of time at 37C~ The samples were removed from the vials and dialyzed against distiLled water in Spectropor* membranes (6000 to 8000 molècular-weight pore size) (Spectrum Medical Industries, IncO, Los Angeles, Cali-fornia) for 48 hrs.

. . .
~mino Acid Analysis Two-milligram samples of AgB and photooxidized anti-: .
- gen B (OX-AgB) (0.5 hr~, 1 hr., and 2 hr. photoo~idation) - were lyophilized to dryness in Pyrex* digestion tubes (13 x ~-.
100 mm). Each sample was re-suspended in 5.7 N HCl and treated with nitrogen gas: the gas was bubbled into each tube for 10 min.; then each tube was sealèd by an oxygen torch and heated at 110C for 20 hrs. Amino acid analysis ~ .
of each sample was performed by the method of Spackman et al ~Anal Chem., 30, 1190, 1958) ~ith a Beckman* model 120C amino i - acid analyzer. The tryptophan content was determined by the method of Spies and Chambers ~Anal. Chem., 21, 1249, 1949).

The results are set forth in Table 1 * - Trade mark CY/~h~

~3~7~733 TABLE I ~ EF'FECT OF PHOTOOXIDATION UPON THE AMINO ACID
CO~lPOSITION OF ANTIGEN B

Amlno Acid Residue Native AgB _ OX-A~B
~M/mole)0.5 hr. 1 hr. 2 hr.

Aspartic 7 7 7 7 Glutamic 8 8 3 8 Threonine 5 5 5 5 Serine 4 4 4 4 Proline 4 4 4 4 Glycine 3 3 ~ 3 3 Alanine 14 14 14 14 Valine 5 ~ 5 5 5 Methionine 1 .5 trace trace Isoleucine 2 2 2 2 Leucine 4 4 4 4 Phenylalanine 3 3 ~3 3 . . , Tyrosine 2 0 0 0 ~`~ Trypt~phan 6 3 3 3 Lysine 6 6 6 6 Histidine 1 1 1 1 Total number o~
residues 75 69.5 69 69 _ e numbers represen~ the ~Moles of amino acid and are rounded - mole of prote1n off to the ne~rest whole integer.
Antisera Rabbit anti-WST serum, prepared as previously described (Malley et al, ~ , 1967), contained l.l mg of antibody pr~tein/ml directed against AgB. Human reaginic serum directed against timothy pollen antigens was obtained from 50 patients and pooled (5 ml from each)~ This pool had a Prausnitz-Kuestner ~P-K) titer of 1:2500.
Precipitation Analysis Rabbit anti-WST serum (0.5 ml~ was added to 0.5 ':

cg/~

~ ~37078 ml of varying concentrations (.03 to 1 mg of protein) of either AgB or OX-AgB. The mixtures were incubated at 37 C
for 30 min. and at 4C for 48 hrs. The precipitates were washed three times with 0.01 M cacodylic saline bu~fer (pH 7) and air-dried; the dried precipitates were quanti~ied as described by Campbell et al., supra, pO 1360 The protein content of each precipitate was determined by nesslerization ~Campbell et al, supra, pg. 53). The results are depicted in Fig. 1. The values reported repxesent the mean of duplicate assays.
Passive Transfer Tests Diluted pooled serum (0.1 ml) was intradermally injected at various sites into the skin of a nonallergic volunteer, and 48 hrs. later each site was challenged with 0.025 ml of AgB (0.1~g of protein/ml)~ Reactions were read 30 min. later and were graded as follows: negative, -;

5- to 10-mm wheal, +; 10- to 15mm whe~l, 2+; 15- to 20-mm wheal, 3+; and greater than 20-mm wheal, 4+
.~ ~
The results are set forth in Table 2 TABLE 2 -- PASSIVE: TRANSFER REACTI~7ITY
OF NATIVE AND PE~OTOOXIDI ZED ~ `
` ANTI&EN B WITH A POOL OF ~` `
:~ ` HUMAN ANTITIMOTHY SERA

Protein P-K Reactions con.
Antigen ~g/ml) 1:100 1:200 1:400 1:800 1:1000 AgB 0.1 4-~ 4+ 3+ 3+ 2+

ox-AgBb 10.0 ~ ~ ~

A serum pool from 50 patients sensitive to timothy grass ; pollen was used in these experiments; the average P-K tlter ~ of this serum was 1:2500. Serum diluted (0.1 ml) was intra~
,, ~
~ 30 dermally injected at various sites into the skin of a .
nonallergic human volunteer. Each site was challenged with .025 ml of antigen 48 hrs. la~er.
.
bThe l-hr. and 2-hr samples.
1~
cg/.,,~c ~.~3'~7l5~
_adioallergosorbent Inhibltlon Two grams of ~hatman* 3 MM paper disks (3 mrn in~
diameter) were activated with CNBr by the method o March et al. (Anal. Biochem., 60, 149, 1974). The ac-tivated disks were washed with 500 ml of cold 0.1 M ~aHCO3-saline buffer ~pH 9) on a sintered glass filter, and the moist disks were added to 30 ml of a.WST solution (1 mg v~ pro-tein/ml) in 0.1 M NaHCO3 saline buffer ~pH 9). This mix- :
ture was stirred for 36 hrs a-t 4C and washed with 500 ~1 each of the following cold reagents: 0.2.M ~a2CO3, 2 M urea, 0~2 M NaAc, and 0.1 M phosphate-buffered saline, pH 7.2.
The WS~-coated di`sks were stored in phosphate-buffered saline at 4 C~ until used.
One-tenth of a milliliter of human reaginic serum diluted 1:37.5 (P-K titer of 10,000~ from a timothy-sensitive patient was incubated with 0.1 ml of varying concentrations .
(0.5 to 20 ~g of ~rotein) of either AgB or OX-AgB for 45 min. at 37 C. The WST-coated disks were.blotted~dry, . and one disk was added to each mixture of preincubated reagin~After 6 hrs~ of incubation at room temperature, each disk was ;:
washed three times (3 x 5 ml), and 5000 to 8000 cpm o 25I~
anti-IgE was added to each disk. The mixtuxes were incuba-ted ; for 16 hrs.at 4C. The WST-coated disks wer~ washed four - times (4 x 5 ml) with RAST buffer and counted by a P~ckard*
scintillation counter ~Packard Instrument Companyr Inc., Downers Grove~ ). Twenty to thirty percen~ o the 5I-anti-IgE added was bound to disks when the reaginic serum ~.
was preincubated with normal human serum~ The percen~ of inhibitation achieved by AgB and OX-AgB was obtained with .
.~ 30 the following equation: ;~
Percent of = (1 RAST counts with added fraction `.
) x 1 00 Inhibition RAST counts uithout fraction : * - Trade mark .~ .
_ 9 _ cg/~1~

~37~7~

The results are depicted in Fig. 2. The values reported represent the means of duplicate assays~
Immunizations . _ LAFl mice (Jackson Laboratories, Bar Harbor, Maine) were immunized with WS~' as previously described ~Fairchild et al., J. Immunol. 115~ 446, 1975). The immunogenicity of OX AgB was studied by il~munization of LA~l mice with OX-AgB (10 to 100 ~g)~ adsorbed to aluminum hydroxide (Levine et al., ~ , 39, 156~ 1970).
Animals were immunized with QX-AgB~at weekly intervals for 2 weeks and given another boost with OX-AgB 3 weeks later. Blood samples were collected 7 days after the initial injectionsl 7, 10, and 14 days a~ter the second injections;
and 7, 10, and 14 days after the final injections of OX-AgB. The serum IgGl and IgE titers of immunized mice ; -were measured by passive cutaneous anaphylaxis as previously . ::
descirbed (Fairchild et al, supra). The results are depicted in Fig. 3. The titers reported represent the mean value - obtained in at least two animals with a pool of serum from at least 5 mice. The arrows indicate when the mice were immunized with AgB or OX-AgB.
Photooxidation converted the yellow-pigmented AgB
to a colorless material with a corresponding decrease in the optical density~at ~80 nm. A comparison of the amino acid composition of AgB and several OX-AgB samples (Table 1) shows a complete reduction in the tyrosine content of OX-AgB and a decrease in the methionine and tryptophan ~- contents; however, the other amino acids remained relatively unchanqed, a fact that suggests the polypeptide structure of AgB was not significantly altered by photooxidation~

The effect of photooxidation upon the antigenic determinants of AgB was evaluated by comparing AgB and OX-AgB for their ability (1) to precipltate rabbit antibody cg/~

3~ 8 against AgB, ~2) to lnitiate P-K reac-tions in ~kin of a nonallergic individual passively sensitized with Ig$-con~
taining serum from timothy-sensitive patients, and (3) to inhibit specific IgE bindirlg to WST-coated paper disks in the ~ST assay.
Rabbit anti-timothy serum containin~ 1..1 mg of antibody protein/ml against AgB failed to yield precipitable antibody with OX-AgB (0.5~hr~, l-hr., and 2-hr.. samples) (Fig. 1).
Table 2 shows that AgB (2.5 ng~ induced strons P-K reactions even at a I:1000 dilution of the reagin pool.
In contrast, a concentration of OX-AgB (1-hr. an.d 2-hr..
samples) 100 times greater ~250 ng~ than the AgB concentration used failed to initiate allergic skin reactions in the skin ::~ of the nonallergic volunteer.
-The ability of OX-AgB to inhibit binding of `~
- 125I anti-IgE with timothy reagin was ~urther evaluated by comparing AgB and OX-AgB inhibition of binding in a RAST
assay for timothy reaginic antibodies tFig~ 2)o In these studies, AgB gave a linear dose-dependent inhibition of 20- 1~5I-anti-IgE binding to IgE-bound WST-coated paper disksr .~ .
but OX-AgB (l-hr~ and 2-hr. samples) failed to inhibit 5I-anti-IgE ~inding even at a 20 ~g concentration. In contrast, urea-treated AgB gave a dose-dependent inhibition of I-anti IgE-bound WST~coated disks identlcal to that of ` native AgB.
~ Anoth~r way to evalua-te the effect of photooxidation :~ upon the antigenic determinant of AgB is to compare the :
immunogenic properties of native and OX-AgB. LAF~ mice in groups of five animals were immunized with either 10 ~g of AgB protein or 10 to 100 ~g of OX-AgB protein (l-hr~ sample).

Mice immunized with AgB made significant levels of IgGl and IgE antibody after a second and third injection of AgB. In :, ~
~ contrast, mice immunized by the same immunization schedule ~,' cg/~

. ~37~7~ :~
with 10 or 100 ~Iy of OX-AgB protein did not produce either IgGl or IgE antibody that reacted with AgB or OX-AgB (Fig. 3).
Antigen-induced lymphocyte transformation was used to compare the relative T-cell~activating properties of AgB and OIY AgBO See Fig. 4 wherein the reported values represent the mean of quadruplicate assays (individual assays vary less than 10~ from the means). Over the con~
centration range tested ~0.1 to 10 ~gj o~ protein), AgB
gave a dose dependent increase in Ag-induced proliferation of immune LAFl mice spleen cells. On the other hand, OX-AgB~
(l-hr. to 2-hr~ samples) had a peak response at 1 ~g that apparently plateaued at ~hi5 level over tha remainder of the ;~
concentrations of OX-AgB tested. These data indicate that OX-AgB retains a signiflcant amount of Ag-induced proli~erative capabilities of native AgB.
Photooxidation of AgB results in a complete loss in the ability of OX~AgB to react With either rabbit anti-AgB
(Fig. 1) or human I~ antibodies (~Table 2, Fig. 2~ directed against the antigenic determinants on AgB ~omple~e acid .
hydrolysis of OX-AgB indicated that the only significant modification of native AgB was -to the quercitin and tyrosine moieties (Table 2). Attempts to modify the antigenic deter-minant of AyB by denaturation with 8 M urea were unsuccessful since native and urea-txeated AgB had identical dose-response curves for 1 5I-anti-IgE binding -to IgE- bound anti~en-`~ conjugated paper disks (Fi~. 23. These data, taken together with data from earlier studies (Malley et al., 1975a, 1975b, -1978) suggest that the major part of the antigenic determinant of AgB is not dependent upon its -three-dimentional polypeptide structure, but is dependent upon the presence of the pigment quercitin in the antigen molecule.
A number of studies (Takatsu et al~, Cell Immunol., 20, 276, 1975; Scibienski et alO, Jo Expo Med., 136, cgj ~ 'Y'`~ ' 1~3~(~7~
1308, 1972; Turkin et al., Proc. Natl. Acad~ Sci., 74, 3~84, 1977) have suggested that de-terminants recogniæed by'T and B
cells may differ~ The B cell receptors combine with haptenic determinants, and T cell receptors are activated by interaction with carrier determinants on the antigen. The above example clearly indicates that OX-AgB neither combines with IgGl or IgE antibodies nor induces the production of antibody against AgB, a fact that suggests OX-AgB does not prime B cells specific for the native antigen. Pre~ious studies ~Fairchild et al., J. Immunol., 117, 2137, 1976) indicated that the'AgB-induced proliferative response of mice '~
immunized with AgB was due to both T and B cells. The carrier ' determinant on OX-AgB retains a substantial portion (~ 40%) of the proliferatl~e response of immune spleen c~lls to AgB ~' (Fig. 3). The aboye example indicates that -the carrier determinant of OX-AgB activates T cells primed with nati~e ' AgB.
: ~, ~ Example 2 - ~ , The following procedure may be employed to ;~
oxidatively modify an antigen with chloramine T:
Protein (1 to 5 mg) is dissolved in 4 ml of 0.05 M phosphate buffer, pH 7. The protein solution is placed ~- in a 30 ml beaker and stirred by a magnetic stirrerO The '~' beaker is placqd in a large plastic dish filled with ice to keep the reactants cold.' Chloramine T (100 ~ to 5 mg3 dissolved in .05 M phosphate buffer, pH 7, is added dropwise and mixed for varying periods of time. To stop the reaction an equal weight of sodium metabisul~ite is added . , to neutralize any remaining oxidizing agent. Excess re~

agents are removed by passage of the solutîon over a Sephadex* G25 column equilibrated in H20 The breakthrough peak' containing the protein is collected and lyophilized.

* - Trade mark ~ 13 - ~' Cs/~ ~

. .

~37a~7~
Ex~mple 3 The followin~ procedure may be utilized to oxidatively modify an antigen with lactoperoxidase. ; ;;
Protein tl to 5 mg) is dissolved in 4 ml of 0.01 M phosphate-buffered saline, pH 7.2. 200 ~g of lacto-peroxidase, and 25 ~1 of 0.03 to 0.1% H2O2 are added.
The reaction mixture is incubated at 37C for 10 to 20 minutes~ during which time two more 25 ~1 aliquots of H202 were added. The reaction was terminated ~y the addition oE 10 volumes of cold 5mM L-cysteine-HCl in phosphate-: :~
buf~ered saline. The protein was reco~ered by passage ofthe mixture o~er a Sephadex* G75 column. ~he enzyme is not held up by the Sephadex* G75 while anti~en Bis retarded by the matrix. Elution of this material is accomplished by continued washing of the column with buffer. The eluted protein is concentrated by lyophilization.

:~.
. ' ` ' ' ' ` , `
... .

.. - ~

, * - Trade mark ~ ' , ~ 14 -cg/;~

Claims (12)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for modifying the antigenic properties of a quercitin moiety containing grass pollen antigen or fragment thereof having the structure:

wherein: G represents glucose, and T represents threonine or a peptide linked to said structure through a threonine molecule;
comprising subjecting said antigen to oxidative conditions such that the said dihydroxy aromatic rings undergo re-arrangement and ring scission to yield a product substan-tially devoid of antigenic properties but capable of ac-tivating T-cells and inducing TS cells.

2. The method of claim 1 wherein said grass pollen antigen is antigen B, antigen D, antigen D1, antigen D2, or antigen D3.

3. The method of claim 1 wherein said oxidative conditions also result in the rearrangement and ring scission of the hydroxy aromatic ring present in the tyrosine moiety of said peptide T.

4. The method of claim 1 wherein the extent of said oxidation is sufficient to result in a modified antigen incapable of reacting with antibodies directed against the antigenic determinant structure of said starting antigen.

5. The method of claim 4 wherein said oxidative conditions are such that substantial modification of the amino acid content of said antigen is avoided.

6. The method of claim 1 wherein said grass pollen is photooxidized.

7. The method of claim 6 wherein said photooxidation is effected by saturating an aqueous solution of said antigen with oxygen and then exposing said solution to light.

8. The modified grass pollen antigen when produced by the process of claim 1.

9. The modified grass pollen antigen when produced by the process of claim 2.

10. The modified grass pollen antigen when produced by the process of claim 5.

11. The modified grass pollen antigen when produced by the process of claim 6.

12. The modified grass pollen antigen when produced by the process of claim 7.